Hell and Wichmann in U.S. Pat. No. 5,731,588 and Baer in U.S. Pat. Nos. 5,777,342, 5,866,911, 5,952,668 and 6,259,104 proposed using stimulated emission to quench excitation in the peripheral parts of an excited spot in a scanning fluorescence microscope to improve resolution. In this method a spot was excited by a light pulse of one wavelength (the excitation wavelength) and quenched by a subsequent light pulse of a second wavelength (the quenching wavelength). The technique is often referred to as “STED” for “stimulated emission depletion” microscopy. This method has been experimentally verified as published in various papers from the laboratory of Stefan Hell.
Other techniques related to STED have been proposed that do not involve stimulated emission, but share with STED the need for providing an exciting beam to the specimen paired with a second beam to reduce excitation in the peripheral parts of the excited spot or line, thereby “sculpting” the excited spot or line to have a sharper central maximum. An example of such alternative techniques, which will be referred to herein as “STED-like,” is excited state depletion microscopy (Hell and Kroug, Appl. Phys. B. 60:495(95)), where the sculpting beam pulse occurs before the excitation pulse and renders molecules exposed to the sculpting beam temporarily insensitive to subsequent excitation by the excitation beam. Furthermore, various STED-like techniques have been proposed that employ proteins and dye molecules able to switch between a fluorescent and non-fluorescent forms by light pulses of appropriate wavelengths.
Although these STED and STED-like techniques promise greatly improved resolution, the requirement of providing two extremely short pulses of different wavelengths in the specimen, such that the pulses are synchronized with each other, has impeded the acceptance of the technique as a practical route to superresolution. Although various proposals have been made to create two synchronized pulsed output beams with different wavelengths from a single input laser (e.g., U.S. Pat. No. 5,866,911), such proposals require some frequency converting devices such as frequency multiplying crystals or optical parametric oscillators to convert the wavelength of one or both of these beams, so that even though the two beams had the same wavelength when they leave the laser, after conversion, when they reach the specimen, the beams had different wavelengths. Besides adding cost and complexity to the instrument, some frequency conversion devices are designed to be optimum at just one wavelength, limiting the versatility of the system. Furthermore the techniques require that the focusing optics be achromatized in order to deal with different excitation and quenching wavelengths. Another factor that has impeded the wider adoption of the technique is that because of the complexity of the apparatus to provide two synchronized pulses of different wavelengths to the specimen, the implementations have been in the form of special purpose integrated instruments not well suited to the modular architecture of current scanning microscopes.